The long-term goal of our research is to understand the roles and the cellular and molecular mechanisms of heparan sulfate proteoglycans (HSPGs) in vascular biology and related human diseases. Angiogenesis is a process of forming new blood vessels and is essential for development and for some common human diseases. HSPGs have been hypothesized to critically regulate angiogenesis. We tested this hypothesis in vivo by endothelial cell (EC)-specific ablation of the heparan sulfate (HS) biosynthetic gene N-deacetylase/N- sulfotransferase-1 (Ndst1) in mice (T-Ndst1-/- mice). We observed that the T-Ndst1-/- mice display angiogenesis defects, showing the first in vivo evidence that EC HS regulates physiological angiogenesis. More intriguingly, we found that the localized angiogenesis defect in T-Ndst1-/- diaphragm leads to congenital diaphragmatic hernia, which phenocopies the developmental defect in the axon guidance molecule Slit3 null (Slit3-/-) mice. This observation suggests a potential mechanism by which EC Ndst1 deficiency may impair the biological function of Slit3, leading to the Slit3-like phenotype in the T-Ndst1-/- mice. Our studies further found that: 1). Slit3 is a novel angiogenic factor; 2). T-Ndst1-/- and Ndst3-/- mice exhibit similar localized angiogenesis defect in diaphragm; 3). EC Ndst1 interacts genetically with Slit3, Robo1 and Robo4 in vivo; 4). Endothelial Ndst1 facilitates Slit3-induced angiogenesis in vitro and in vivo; and 5). HS binds both Slit3 and Robo1. These findings led to our central hypothesis that EC HSPG interacts with Slit/Robo to regulate angiogenesis. To test this hypothesis, we will pursue the following three specific aims: Aim. 1. Characterize the function of the Slit3/Robo pathway in angiogenesis. We will examine the vascular pattern and retinal vascularization in Slit3 and Robo mutant mice to determine the requirement of Slit3/Robo for developmental angiogenesis. We have observed that Slit3 induces neovascularization in vivo and will further determine the Robos that Slit3 interacts with to regulate angiogenesis. We will also determine the effects of the Slit3-Robo interaction on EC functions. Aim 2. Determine the regulatory role of EC HS on Slit3/Robo-mediated angiogenesis. We have observed that EC Ndst1 facilitates Slit3-mediated angiogenesis in vivo and will further determine whether Robos are similarly regulated. We will continue the breeding of T-Ndst1-/- mice with Slit3/Robo mutant mice to determine if EC Ndst1 interacts genetically with Slit3/Robo. We will also determine the consequences of Ndst1 deficiency on Slit3/Robo-mediated EC function and Slit3/Robo binding. Aim 3. Elucidate the structural features of Slit3/Robo-binding sites within HS. We will produce recombinant human Slit3, Robo1 and Robo4 proteins and prepare affinity columns to isolate Slit3-, Robo1- and Robo4-binding HS fragments. The structural features of Slit3/Robo-binding HS fragments will be deduced by size and composition analyses in conjunction with HS analogs. PUBLIC HEALTH RELEVANCE: Angiogenesis is critically related to human diseases, such as cancer and tissue repair. Therefore, our proposed studies are expected to not only significantly advance our understanding of angiogenesis, but may also lead to novel pharmaceutical interventions for treatment of angiogenesis-based human diseases.